Lipid metabolites including prostaglandins, leukotrienes, and platelet activating factor (PAF) are produced during parturition. Both fetal and maternal membranes are implicated in this production. Understanding th metabolic pathways involved in eicosanoid production by these tissues is important in elucidating the steps involved in the initiation of both normal and preterm labor. The aim of this project is to determine the mechanisms of prostaglandin control in a well-defined cell line system where the results can eventually be applied to the more complex case of fetal and decidual membranes. The putative control step in the generation of prostaglandins is the release of arachidonic acid from the sn-2 position of membrane phospholipids by phospholipase (PLA2). Thus, our approach will be to identify and characterize the phospholipases responsible for releasing arachidonic acid and for controlling prostaglandin production. This involves isolating these enzymes, characterizing them in vitro, and correlating their characteristics with those of prostaglandin production in the cell. Toward this end, our laboratory has identified various signal transduction mechanisms related to prostaglandin control in the transformed macrophage-like cell line, P388D1. We have succeeded in separating and purifying several phospholipases from these cells, including a novel Ca2+-independent PLA2 whose activity is regulated by ATP and possibly by a regulatory protein. During the next grant period, we will further characterize this Ca2+- independent PLA2. We will also continue our studies of a secretory PLA2 which we have found on the exterior of P388D1 cells and which is similar to the PLA2 associated with preterm labor in patients with bacterial infections. Antisense RNA techniques will also be utilized to inhibit the production of specific phospholipases in the cells to evaluate their role in arachidonic acid release and prostaglandin production. We will also continue exploring chemical inhibitors for their ability to inhibit both isolated enzymes and prostaglandin production in the cells. In addition to these phospholipase studies, we will also explore the role of the inducible cyclooxygenase II enzyme. We will continue to characterize the signal transduction mechanisms and prostaglandin production in the macrophage cells as well as in the human amnionic WISH cell line and in cultured primary amnionic cells. PLA2 in the latter cells will also be identified and studied.